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United States Patent |
6,225,124
|
Houwen
,   et al.
|
May 1, 2001
|
Diluting reagent and method compelling time-independent consistency in MCV
assay
Abstract
Aqueous blood-sample diluting reagent and method of its use for compelling
a morphological change in a blood sample to yield an MCV value assayed at
elapsed time after the sample is drawn to be consistent within a
diagnostically acceptable range with the original, immediate post-drawing
MCV value. Selection of a small amount of a predetermined surfactant added
within a limited range of concentration, and of a salt for adjusting
osmotic pressure of the sample is thereby determined. The blood sample is
treated with an anti-coagulant agent immediately post-drawing, and for
assay in a particle analyzer at post-drawing elapsed time is diluted with
the reagent solution. The reagent has an osmotic pressure (.pi.) of
approximately 150-400 mOsm/kg and a pH of 6.0-8.5. The surfactant is
present in a 0.0005% to 0.5% concentration and has a hydrophile-lipophile
balance (HLB) of 10-20.
Inventors:
|
Houwen; Berend (Redlands, CA);
Uchihashi; Kinya (Kakogawa, JP);
Hamaguchi; Yukio (Akashi, JP);
Mast; Rolf (Riverside, CA)
|
Assignee:
|
Sysmex Corporation (Kobe, JP)
|
Appl. No.:
|
323689 |
Filed:
|
June 2, 1999 |
Current U.S. Class: |
436/63; 252/408.1; 435/2; 436/8; 436/10; 436/18; 436/176; 436/179 |
Intern'l Class: |
G01N 033/48 |
Field of Search: |
436/8,10,18,63,70,174,176,179
435/2
252/408.1
|
References Cited
U.S. Patent Documents
4299726 | Nov., 1981 | Crews et al.
| |
4506018 | Mar., 1985 | North, Jr. | 436/10.
|
5116539 | May., 1992 | Hamaguchi et al. | 252/408.
|
5413938 | May., 1995 | Tsujino et al. | 436/63.
|
5496734 | Mar., 1996 | Sakata | 436/63.
|
5618733 | Apr., 1997 | Sakata et al. | 436/17.
|
5888752 | Mar., 1999 | Malin et al. | 435/7.
|
Foreign Patent Documents |
1-33780 | Jul., 1989 | JP.
| |
7-82010 | Sep., 1995 | JP.
| |
8-33388 | Mar., 1996 | JP.
| |
8-122327 | May., 1996 | JP.
| |
Other References
English abstract of JP96033388, Mar. 29, 1996.*
English abstract of JP08122327, May 17, 1996.
|
Primary Examiner: Wallenhorst; Maureen M.
Attorney, Agent or Firm: Shinjyu Intellectual Property Firm
Claims
What is claimed is:
1. A blood-sample diluting reagent for compelling a volume-stable
erythrocyte morphology in a mean corpuscular volume (MCV) assay of drawn
blood samples, said reagent being an aqueous solution comprising:
(a). at least one nonionic surfactant present in the aqueous solution in a
5.0.times.10.sup.-4 to 5.0.times.10.sup.-1 % weight concentration and
having a hydrophile/lipophile balance of 10-20, said at least one nonionic
surfactant selected to be completely soluble in water to yield a clear
solution at a minimum weight concentration of 0.1%, wherein said at least
one nonionic surfactant is selected from the group consisting of:
##STR3##
wherein R indicates one of an alkyl chain, an alkenyl chain and an alkynyl
chain, respectively having 12-24 carbons; and n, n.sub.1 +n.sub.2
+n.sub.3, and n.sub.1 +n.sub.2 +n.sub.3 +n.sub.4 +n.sub.5 +n.sub.6
indicate an integer 5-70; and
(b) a salt for adjusting osmotic pressure of the aqueous solution to be
approximately 150-400 mOsm/kq; whereby
the at least one nonionic surfactant and the salt are selected so that
discrepancy between MCV values of a freshly drawn blood sample not treated
with said reagent and measured immediately post-drawing, and of a freshly
drawn blood sample immediately treated with said reagent yet measured up
to 72 hours or more post-drawing, is restricted to be within a
diagnostically acceptable range.
2. A blood-sample diluting reagent as set forth in claim 1, wherein the
osmotic pressure is adjusted to be approximately 230-350 mOsm/kg.
3. A blood-sample diluting reagent as set forth in claim 2, wherein the
osmotic pressure is adjusted to be approximately 260-320 mOsm/kg.
4. A blood-sample diluting reagent as set forth in claim 1, wherein said at
least one nonionic surfactant is. polyoxyethylne (20) oleyl ether.
5. A blood-sample diluting reagent as set forth in claim 1, wherein said
diluting reagent has a pH of 6.0-8.5.
6. A method for mean corpuscular volume (MCV) assay of a drawn blood sample
preserved for up to at least 72 hours, the method comprising:
a step of treating said drawn blood sample with an anti-coagulant agent;
a step of diluting said anti-coagulant treated blood sample with a diluting
reagent as set forth in claim 1 which compels a morphological change in
erythrocytes of said blood sample to yield an MCV value at post-drawing
elapsed time to be consistent within a diagnostically acceptable range
with immediate post-drawing MCV value of said blood sample; and
a step of assaying said blood sample to obtain its MCV value in a particle
analyzer.
7. A method for MCV assay as set forth in claim 6, wherein in said step of
assaying the blood sample, particle analyzer employs an electrical
resistance mechanism.
8. A method for MCV assay as set forth in claim 7, wherein the particle
analyzer assays by a sheath flow electrical resistance mechanism.
9. A method for MCV assay as set forth in claim 6, wherein the at least one
nonionic surfactant in the diluting reagent is polyoxyethylene (20) oleyl
ether.
10. A method for MCV assay as set forth in claim 6, wherein the diluting
reagent has a pH of 6.0-8.5.
Description
BACKGROUND OF THE INVENTION
1. Technical Field
The present invention relates to assaying mean corpuscular volume (MCV) in
blood samples; more particularly the invention relates to reagents for, as
well as a method of use in, MCV assays using a particle analyzer.
2. Description of Related Art
Automated hematological analyzers are now widely used in the area of
clinical examination. As devices for screening patients in medical
diagnostic procedures, they are designed for rapid analysis of blood
constituents. Such devices can perform the multiple assays of a complete
blood count (CBC), including such items as red blood cell (RBC) count,
white blood cell (WBC) count, leukocyte classification, hemoglobin
concentration (Hb), hematocrit (Ht) and platelet (PLT) count.
The MCV is one among vital items obtained by calculating from these
cytometric assay values. The MCV is obtained as the hematocrit divided by
the RBC count, that is, the value Ht/RBC, the hematocrit being the
percentage of erythrocytes (red blood cells) in a unit volume of whole
blood. The MCV, then is the average volume of a single red blood cell, and
accordingly it is given in fL, femtoliters, or 10.sup.-15 L.
The hematological analyzers noted above employ flow cytometers. In flow
cytometers, a diluted blood sample is discharged into a flow cell at high
speed through a very thin nozzle, and the discharging flow from the nozzle
is surrounded by the cylindrical flow of a sheathing liquid (laminar
flow). By narrowing the laminar flow, the sample flow can thus be focused,
to the point at which the flow is essentially a cell-by-cell linear
succession. The cells, whose passage in the sample flow is thus controlled
by the laminar flow, are then detected optically, by irradiating the flow
with a laser beam, as well as electrically, by measuring the resistance or
conductivity.
To prepare a whole blood sample for flow-cytometric assay in a
hematological analyzer as described above, the sample generally must be
treated with an anti-coagulant agent such as an EDTA salt, and diluted
with a physiologically isotonic solution.
As diluents for diluting whole blood, there are in general such solutions
as physiological saline, Ringer's solution, Locke's solution, and Tyrode's
solution. In performing the above-described assays in a hematological
analyzer, the foregoing diluents can be used. Ordinarily, however,
diluents optimized for individual analyzers are employed.
A basic problem with the current technology in preparing whole blood
samples for MCV assay is that the actual MCV changes with post-blood
drawing elapsed time. The MCV value that is necessary and important in
clinical diagnosis is that which would be obtained immediately after the
blood sample is drawn, that is, that value which corresponds to the MCV of
the original blood.
In practice, the MCV value as part of a CBC is not measured until as much
as 72 hours have passed following drawing of the blood sample. At that
point, however, blood samples as such no longer yield suitable MCV assay
values. After the blood is drawn, the RBCs in the sample swell, such that
MCV values measured with the post-drawing elapse of time are progressively
larger than the original RBC mean volume. The original MCV is the actual
measurement diagnostically required.
Japanese Pat. Publ. 8-33388 (1996), Japanese Pat. Publ. 7-82010 (1995), and
Japanese Laid Open Pat. 8-122327 (1996) disclose various aqueous solutions
containing nonionic surfactants. Nevertheless, whichever of these
solutions is used as a flow-cytometer sheathing liquid, and the nonionic
surfactants are added for eliminating air bubbles within the flow chamber.
Furthermore, Japanese Pat. Publ. 1-33780 (1989) discloses a diluent into
which a nonionic surfactant has been added to neutralize the effect on
erythrocytes of antiseptic agents, ultimately with the object of
restraining post-dilution change in the measured MCV due to the antiseptic
agents. Nevertheless, Japanese Pat. Publ. 1-33780 does not disclose a
method of restraining change in measured MCV due to the passage time after
drawing blood samples.
It would be an important advance in the art if a means could be found to
bring about consistency with blood sample original values in hematocrit
and RBC count measurements made up to 72 hours after the blood is drawn.
SUMMARY OF THE INVENTION
The present invention compels a blood sample to yield an MCV value assayed
at elapsed time after the sample is drawn to be remarkably consistent with
the original MCV value of the sample, that is, the immediate post-drawing
MCV value.
A reagent in accordance with the invention is a blood sample aqueous
diluting solution that includes a small amount of a predetermined
surfactant added within a limited range of concentration. The osmotic
pressure of the reagent is adjusted with a suitable substance to be within
a predetermined range.
A reagent for the present invention is an aqueous solution including a
nonionic surfactant, and a salt or suitable substance for adjusting
osmotic pressure (.pi.) to be approximately 150-400 mOsm/kg.
Preferably the osmotic pressure (.pi.) of the reagent is approximately
230-350 mOsm/kg; especially preferable is an osmotic pressure (.pi.) of
approximately 260-320 mOsm/kg. As examples of nonionic surfactants in an
embodiment of the present invention, any of the following can be used.
##STR1##
wherein R indicates one of an alkyl chain, an alkenyl chain and an alkynyl
chain, respectively having 12-24 carbons; and n, n.sub.1 +n.sub.2
+n.sub.3, and n.sub.1 +n.sub.2 +n.sub.3 +n.sub.4 +n.sub.5 +n.sub.6
indicate an integer 5-70.
In a preferred embodiment of the present invention, the nonionic surfactant
includes at least one of an alkyl chain, an alkenyl chain and an alkynyl
chain, respectively having 12-24 carbons.
The nonionic surfactant preferably includes a polyoxyethylene chain having
an ethylene oxide molar addition number of 5-70.
As concrete examples, any of the following nonionic surfactants can be used
in an embodiment in accordance with the present invention.
a) Polysorbate-80 (oleate esters of sorbitan plus 20 moles PEG), Croda
Co.'s "Crillet 4."
b) Steareth-20 (Stearyl alcohol+PEG 20), Croda Co.'s "Volpo S-20."
c) PEG-60 Almond Glycerides (Almond mono and diglycerides with 60 moles
PEG), Croda Co.'s "Crovol A-70."
d) PEG-23 Oleate (oleic acid plus PEG23 ester), Croda Co.'s "Crodet 0 23."
One further preferable nonionic surfactant is Oleth-20 (polyoxyethylene
[20] oleyl ether). This surfactant contains a polyoxyethylene oleyl ether
mixture wherein the average number of oxyethylene units per molecule is
approximately 20.
CTFA Names is the source of the names for the substances noted above.
It should be apparent that within the scope of the present invention,
mixtures of nonionic surfactants as herein defined could also be used. It
should also apparent that commercially available nonionic surfactants are
suitable for this invention, provided that they otherwise conform to the
parameters given herein. It is well known that these commercially
available nonionic surfactants are not chemically pure materials.
Consequently, concomitant introduction of related nonionic structures and
extraneous materials into a reagent in accordance with the invention is
within its scope, on the condition that the foregoing parameters are
satisfied.
It is particularly important that the surfactants or mixtures thereof are
transparent in the salt solution. The nonionic surfactants must be
completely soluble in water to yield a clear solution at a minimum weight
concentration of 0.1%.
The concentration of the given nonionic surfactants for inclusion in the
reagent can be suitably determined according to the osmotic pressure and
the composition of the salt solution as the solvent. In general, however,
the nonionic surfactants can be employed at a low concentration in the
range of approximately 0.0005%-0.5%. A preferable concentration is in the
range of approximately 0.001%-0.1%, and most preferably is in the range of
approximately 0.005%-0.05%.
In accordance with the invention, the nonionic surfactant is used in the
salt solution thus designed for diluting and stabilizing blood samples for
hematological assay.
The nonionic surfactant preferably has a hydrophile/lipophile (HLB) balance
of 10-20.
The pH of a reagent in accordance with the invention is preferably 6.0-8.5.
A reagent given by the present invention further includes a buffer for
stabilizing its pH, an additive for adjusting the osmotic pressure of the
reagent solution, an antiseptic substance, or an anti-oxidant. Suitable
buffers are not particularly limited; however, phosphate buffers, borate
buffers, tris buffer, imidazole buffers, etc. are preferable.
Suitable substances for adjusting the pH of the reagent are not
particularly limited, but can be, for example, hydrochloric acid or sodium
hydroxide.
Suitable substances for adjusting for adjusting the osmotic pressure are
not particularly limited. For example, at least one selected from an
alkali metal salt or an alkali earth metal salt of sodium chloride, and
potassium chloride may be used. Alternatively, a sugar such as sucrose,
glucose, etc., or a polyethylene glycol may be used.
A method in accordance with the present invention includes: (a) an
anti-coagulant treatment process wherein the blood sample is treated with
an anti-coagulant agent; (b) a dilution process wherein the blood sample
treated with an anti-coagulant agent is diluted with a reagent solution
containing a surfactant to compel a morphological change in red blood
cells in the sample, and (c) an assay process wherein the blood sample is
assayed in a particle analyzer following the dilution process at the
elapse of, for example, at least 48 hours after blood drawing.
In a preferred aspect of the invention, the hematological assay is
conducted in a particle analyzer based on the electrical resistance
principle; the sheath-flow electrical resistance mechanism is more
preferable.
The assay process can be a process wherein the blood sample is assayed in a
particle analyzer following said dilution process at the elapse of up to
at least 72 hours.
Prior to the assay, whole blood has to be treated with an anti-coagulant
agent.
As a standard anti-coagulant treatment, an appropriate amount of an
anti-coagulant agent such as EDTA salt is added into a whole blood sample
immediately after blood drawing.
The formula of an example anti-coagulant agent is given below.
##STR2##
The samples are preferably obtained by using a vacuum blood-drawing tube,
and preferably are treated with an anti-coagulant agent at the time the
blood is drawn.
A diluting solution of the present invention compels morphological changes
in blood sample RBCs, and acts on blood samples up to at least 72 hours
old accordingly to yield MCV results consistent with those obtained
immediately post-drawing.
The action mechanism of the present invention has not been proved.
Nevertheless, it was discovered that diluting a blood sample with a
solution adjusted to have a predetermined osmotic pressure and containing
a predetermined surfactant compels a morphological change in the blood
sample RBCs that restores consistency in assayed MCV. The morphological
change is to an extent that the MCV value of red blood cells measured at
elapsed time post-drawing is equivalent within a diagnostically acceptable
range to the MCV value immediately after a blood sample is drawn.
Consequently, the present invention provides consistent MCV values
throughout the diagnostic life of the blood sample.
This was corroborated by suspending a freshly drawn blood sample as well as
a 72-hour old sample into a diluting solution reagent in accordance with
the invention, and examining the respective samples microscopically. A
similar level of stomatocytes was observed in both samples.
By employing a reagent in accordance with the invention, time-dependent
changes in the MCV of a blood sample can be limited. Discrepancies in MCV
assayed at different times during the period between when the sample is
drawn, and at least 72 hours thereafter, can be controlled to be within
about.+-.4(fL), which is well within a diagnostically acceptable range.
The foregoing and other objects, features, aspects and advantages of the
present invention will become more apparent from the following detailed
description.
DETAILED DESCRIPTION OF EMBODIMENTS
Embodiment 1
Blood samples were treated with EDTA-3K anti-coagulant. The samples were
then subjected to an MCV assay in Sysmex Co. Ltd.'s automated
hematological analyzer SE-9500 immediately post blood-drawing, and at 72
hours thereafter. The blood samples were stored at 25.degree. C.
Reagent A (Conventional): 15 mM Phosphate Buffer Solution
The osmotic pressure of this reagent was adjusted with sodium chloride to
be approximately 250 mOsm/kg, and
Reagent B: 15 mM Phosphate Buffer Solution; 0.015% Polyoxyethylene (20)
Oleyl Ether
The osmotic pressure of this reagent was adjusted with sodium chloride to
be approximately 320 mOsm/kg, and its pH was adjusted with sodium
hydroxide to be approximately 7.8.
MEAN CORPUSCULAR VOLUME IN FEMOTOLITERS (fL)
Post Blood-Drawing Time
Reagent Immediate 72 hours Variation Percentage
SAMPLE NO. 1
Reagent A 83.8 (fL) 97.1 (fL) +13.3 (fL) +15.9 (%)
Reagent B 88.2 (fL) 90.4 (fL) +2.2 (fL) +2.5 (%)
SAMPLE NO. 2
Reagent A 89.7 (fL) 103.9 (fL) +14.2 (fL) +15.8 (%)
Reagent B 93.1 (fL) 96.0 (fL) +2.9 (fL) +3.1 (%)
SAMPLE NO. 3
Reagent A 89.6 (fL) 103.9 (fL) +14.3 (fL) +16.0 (%)
Reagent B 93.9 (fL) 96.0 (fL) +2.1 (fL) +2.2 (%)
Embodiment 2
Blood samples were treated with EDTA-2K anti-coagulant. The samples were
then subjected to an MCV assay in Sysmex Co. Ltd's automated hematological
analyzer SE-9500 12 hours and 48 hours post blood-drawing. The blood
samples were stored at 25.degree. C.
Reagent A: 15 mM Phosphate Buffer Solution
The osmotic pressure of this reagent was adjusted with sodium chloride to
be approximately 250 mOsm/kg, and its pH was adjusted with sodium
hydroxide to be approximately 7.8.
Reagent C: 15 mM Phosphate Buffer Solution
The osmotic pressure of this reagent was adjusted with sodium chloride to
be approximately 285 mOsm/kg, and its pH was adjusted with sodium
hydroxide to be approximately 7.8.
Reagent D: 15 mM Phosphate Buffer Solution; 0.015% Polyoxyethylene (20)
Oleyl Ether The osmotic pressure of this reagent was adjusted with sodium
chloride to be approximately 285 mOsm/kg, and its pH was adjusted with
sodium hydroxide to be approximately 7.8.
MEAN CORPUSCULAR VOLUME IN FEMOTOLITERS (fL)
Post Blood-Drawing Time
Reagent 12 hours 48 hours Variation Percentage
Reagent A 93.5 (fL) 102.6 (fL) +9.1 (fL) +9.7 (%)
Reagent C 93.4 (fL) 103.7 (fL) +10.3 (fL) +11.0 (%)
Reagent D 92.7 (fL) 95.4 (fL) +2.7 (fL) +2.9 (%)
The results indicate that the increase in osmotic pressure of the diluent
did not have control over the variation in MCV of the blood samples with
post-drawing elapse of time. The addition of nonionic surfactant to the
diluent, however, does demonstrate a distinct effect in holding down the
variation in MCV.
Embodiment 3
In Embodiment 3, variation in MCV was measured employing reagents prepared
from Reagent D by varying its osmotic pressure. The osmotic pressures of
the reagents were adjusted by changing the amount of sodium chloride
added. The blood samples was stored at 25.degree. C. The samples were
assayed in Sysmex Co. Ltd.'s automated hematological analyzer SE-9500 to
determine MCV.
Reagent D: .pi. = 285 mOsm/kg (pH 7.8)
Reagent E: .pi. = 250 mOsm/kg (pH 7.8)
Reagent F: .pi. = 268 mOsm/kg (pH 7.8)
Reagent G: .pi. = 300 mOsm/kg (pH 7.8)
Reagent H: .pi. = 320 mOsm/kg (pH 7.8)
MEAN CORPUSCULAR VOLUME IN FEMOTOLITERS (fL)
Post Blood-Drawing Time
Reagent 12 hours 48 hours Variation Percentage
Embodiment 3-1
Reagent A 94.5 (fL) 103.7 (fL) +9.2 (fL) +9.7 (%)
Reagent E
(.pi. 250) 93.4 (fL) 98.3 (fL) +4.9 (fL) +5.2 (%)
Reagent D
(.pi. 285) 92.3 (fL) 94.2 (fL) +1.8 (fL) +2.0 (%)
Embodiment 3-2
Reagent A 97.4 (fL) 106.7 (fL) +9.3 (fL) +9.5 (%)
Reagent F
(.pi. 268) 95.4 (fL) 99.0 (fL) +3.6 (fL) +3.8 (%)
Reagent D
(.pi. 285) 94.1 (fL) 94.3 (fL) +0.2 (fL) +0.2 (%)
Embodiment 3-3
Reagent A 94.5 (fL) 103.4 (fL) +8.9 (fL) +9.4 (%)
Reagent D
(.pi. 285) 92.3 (fL) 95.9 (fL) +3.6 (fL) +3.9 (%)
Reagent G
(.pi. 300) 91.6 (fL) 95.0 (fL) +3.4 (fL) +3.7 (%)
Reagent H
(.pi. 320) 91.6 (fL) 93.5 (fL) +1.9 (fL) +2.1 (%)
The results indicate that variation in MCV could be controlled to be within
.+-.4 (fL) by keeping the osmotic pressure of the reagent in the range of
260-320 mOsm/kg. Within this range, higher osmotic pressure demonstrated
more control over variation in MCV.
Embodiment 4
In Embodiment 4, variation in MCV was measured employing reagents prepared
by adding the nonionic surfactant polyoxyethylene (20) oleyl ether to
Reagent A at various concentrations. The blood samples were stored at
25.degree. C. The samples were assayed in Sysmex Co. Ltd.'s automated
hematological analyzer SE-9500 to determine MCV.
Reagent A: 0.000% nonionic surfactant
Reagent E: 0.015% nonionic surfactant
Reagent I: 0.150% nonionic surfactant
Reagent J: 0.300% nonionic surfactant
MEAN CORPUSCULAR VOLUME IN FEMOTOLITERS (fL)
Post Blood-Drawing Time
Conc. (%) 12 hours 48 hours Variation Percentage
Reagent A 95.8 (fL) 105.9 (fL) +10.1 (fL) +10.5 (%)
(0.000%)
Reagent E 95.4 (fL) 100.0 (fL) +5.6 (fL) +5.9 (%)
(0.015%)
Reagent I 96.6 (fL) 100.9 (fL) +4.3 (fL) +4.5 (%)
(0.150%)
Reagent J 96.2 (fL) 100.4 (fL) +4.2 (fL) +4.4 (%)
(0.300%)
The results indicate that the nonionic surfactant in a wide range of
concentrations was relatively effective for controlling variation in MCV.
Embodiment 5
Reagents in Embodiment 5 were prepared by adding approximately 0.015%
polyoxyethylene (20) oleyl ether to Reagent E, and then adjusting the
osmotic pressure of the diluent to 285 mOsm/kg with the following
substances.
Reagent D: sodium chloride
Reagent K: sucrose
Reagent L: glucose
Reagent M: polyethylene glycol (MW400)
The effect on variation in MCV of the foregoing substances employed for
adjusting the osmotic pressure of the reagents was measured in Embodiment
5. The blood samples were stored at 25.degree. C. The samples were assayed
in Sysmex Co. Ltd.'s automated hematological analyzer SE-9500 to determine
MCV.
MEAN CORPUSCULAR VOLUME IN FEMOTOLITERS (fL)
Post Blood-Drawing Time
Reagent 12 hours 48 hours Variation Percentage
Embodiment 5-1
Reagent A 94.7 (fL) 105.1 (fL) +10.4 (fL) +11.0 (%)
Reagent D 92.5 (fL) 94.8 (fL) +2.3 (fL) +2.5 (%)
Reagent K 91.6 (fL) 94.9 (fL) +3.3 (fL) +3.6 (%)
Reagent L 93.0 (fL) 95.8 (fL) +2.7 (fL) +2.9 (%)
Embodiment 5-2
Reagent A 94.4 (fL) 104.7 (fL) +10.3 (fL) +10.9 (%)
Reagent D 92.8 (fL) 95.6 (fL) +2.8 (fL) +3.0 (%)
Reagent M 92.9 (fL) 93.6 (fL) +0.7 (fL) +0.8 (%)
The substances employed generally for adjusting osmotic pressure are not
limited to those given in Embodiment 5-1 and Embodiment 5-2. The results
indicate, however, that salts, sugars and polyethylene glycol are
preferable.
While several embodiments have been chosen merely to illustrate the present
invention, it will be apparent from this disclosure to those skilled in
the art that various changes and modifications can be made herein without
departing from the scope of the invention as defined by the appended
claims and their equivalents.
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